The surface chemistry of acid pretreated-biomass, used in ethanol production, is different from that found in plant tissues, naturally digested by fungal cellulase enzymes, in two important ways: (1) pretreatment heats the substrate past the phase-transition temperature of lignin; and (2) pretreated biomass contains less acetylated hemicellulose. Thus, it is believed, that the cellulose fibers of pretreated-biomass are coated with displaced and modified lignin. This alteration results in a non-specific binding of the protein with the biomass, which impedes enzymatic activity. Moreover, where the pretreated biomass is a hardwood-pulp it contains a weak net-negatively charged surface, which is not observed in native wood. Therefore, for the efficient production of ethanol from a pretreated biomass such as corn stover, wood or other biomass it is desirable to enhance the catalytic activity of glycosyl hydrolases specifically the cellobiohydrolases.
Trichoderma reesei CBH I (SEQ ID NO: 5) is a mesophilic cellulase which plays a major role in the hydrolysis of cellulose. An artificial ternary cellulase system consisting of a 90:10:2 mixture of T. reesei CBH I, Acidothermus cellulolyticus EI, and Aspergillus niger β-D-glucosidase is capable of releasing as much reducing sugar from pretreated yellow poplar as the native T. reesei system after 120 h. This result is encouraging for the ultimate success of engineered cellulase systems, because this artificial enzyme system was tested at 50° C., a temperature far below that considered optimal for EI, in order to spare the more heat labile enzymes CBH I and β-D-glucosidase. To increase the efficiency of such artificial enzyme systems it is desirable to engineer new T. ressei CBH I variant enzymes capable of active expression in heterologous hosts. The use of the heterologous host Aspergillus awamori, could provide an excellent capacity for synthesis and secretion of T. reesei CBH I because of its ability to correctly fold and post-translationally modify proteins of eukaryotic origin. Moreover, A. awamori is believed to be an excellent test-bed for Trichoderma coding sequences and resolves some of the problems associated with site directed mutagenesis and genetic engineering in Trichoderma. 
In consideration of the foregoing, it is therefore desirable to provide variant cellulase enzymes having enzymatic activity when expressed in a heterologous host, and to provide variant cellulase enzymes that have improve thermal tolerance over the native as produced by Trichoderma reesei. 